CN107338217B

CN107338217B - Differentiation of human embryonic stem cells

Info

Publication number: CN107338217B
Application number: CN201710695469.5A
Authority: CN
Inventors: J.许
Original assignee: Janssen Biotech Inc
Current assignee: Janssen Biotech Inc
Priority date: 2010-05-12
Filing date: 2011-05-11
Publication date: 2021-02-09
Anticipated expiration: 2031-05-11
Also published as: ZA201209383B; EP2569419A4; US9752125B2; US20110281355A1; AR081040A1; CA2800610C; CA2800610A1; MX351515B; CN102884176A; JP7149190B2; KR20180110180A; SG10201503652WA; SG185511A1; WO2011143299A2; BR112012028855A2; CN102884176B; RU2018120334A; RU2663339C1; JP2017012178A; RU2587634C2

Abstract

The present invention provides methods for promoting differentiation of pluripotent stem cells into insulin producing cells. In particular, the invention provides a method of making a population of cells expressing markers characteristic of the pancreatic endoderm lineage, wherein greater than 50% of the cells in the population co-express PDX1 and NKX 6.1.

Description

Differentiation of human embryonic stem cells

The application is a divisional application, the Chinese application number of the parent application is 201180023585.3, the international application number is PCT/US2011/036043, and the application date is 2011, 5 and 11.

Cross reference to related patent applications

This application claims the benefit of U.S. provisional patent application No.61/333,831, filed on 12/5/2010, which is incorporated herein by reference in its entirety for all purposes.

Technical Field

The present invention provides methods for promoting differentiation of pluripotent stem cells into insulin producing cells. In particular, the invention provides a method of making a population of cells expressing markers characteristic of the pancreatic endoderm lineage, greater than 50% of which co-express PDX1 and NKX 6.1.

Background

The advances in cell replacement therapy for type I diabetes and the lack of transplantable islets have focused attention on developing sources of insulin producing cells or beta cells suitable for engraftment. One approach is to generate functional beta cells from pluripotent stem cells, such as embryonic stem cells.

During embryonic development in vertebrates, pluripotent stem cells can give rise to a population of cells comprising three germ layers (ectoderm, mesoderm and endoderm) in a process known as gastrulation. Tissues such as thyroid, thymus, pancreas, intestine and liver will develop from the endoderm, via intermediate stages. The intermediate stage in the process is the formation of definitive endoderm. Definitive endoderm cells can express a variety of markers such as HNF3 β, GATA4, MIXL1, CXCR4, and SOX 17.

Differentiation of definitive endoderm into pancreatic endoderm results in the formation of pancreas. Pancreatic endoderm cells express the pancreatic duodenal homeobox gene PDX 1. In the absence of PDX1, the pancreas did not develop after formation of ventral and dorsal pancreatic buds. Thus, PDX1 expression marks a key step in pancreatic organogenesis. The mature pancreas includes, among other cell types, exocrine and endocrine tissues. Exocrine and endocrine tissues arise from differentiation of the pancreatic endoderm.

Cells with islet cell characteristics were reported to be derived from embryonic cells of mice. For example, Lumelsky et al (Science 292:1389,2001 (Science, vol.292, p.1389, 2001)) reported that mouse embryonic stem cells differentiated into an insulin-secreting structure similar to pancreatic islets. Soria et al (Diabetes 49:157,2000 (Diabetes, Vol. 49, p. 157, 2000)) reported that insulin-secreting cells derived from mouse embryonic stem cells normalize streptozotocin-induced blood glucose in diabetic mice.

In one example, Hori et al (PNAS 99:16105,2002 (Proc. Natl. Acad. Sci. USA, 99, 16105, 2002)) disclose that treatment of mouse embryonic stem cells with a phosphatidylinositol 3-kinase inhibitor (LY294002) produces cells similar to beta cells.

As another example, Blyszczuk et al (PNAS 100:998,2003 (Proc. Natl. Acad. Sci. USA, Vol. 100, p. 998, 2003)) reported that insulin-producing cells were generated from mouse embryonic stem cells that constitutively expressed Pax 4.

Micalalef et al reported that retinoic acid can regulate the orientation of embryonic stem cells to form PDX1 positive pancreatic endoderm. The most efficient way to induce expression of Pdx1 on day 4 of differentiation of embryonic stem cells, i.e., during the period corresponding to the terminal stage of gastrulation of the embryo, was to add retinoic acid (Diabetes 54:301,2005 (Diabetes, Vol. 54, p. 301, 2005)) to the culture.

Miyazaki et al reported mouse embryonic stem cell lines overexpressing Pdx 1. Their results indicate that exogenous Pdx1 expression significantly enhanced the expression of insulin, somatostatin, glucokinase, neurogenin 3, p48, Pax6 and Hnf6 genes in the resulting differentiated cells (Diabetes 53:1030,2004 (vol. 53, p 1030, 2004)).

Skoudy et al reported that activin A, a member of the TGF- β superfamily, upregulates the expression of pancreatic exocrine genes (p48 and amylase) and endocrine genes (Pdx1, insulin and glucagon) in mouse embryonic stem cells. The greatest effect was observed with 1nM activin A. They also observed that the mRNA expression levels of insulin and Pdx1 were not affected by retinoic acid; however, treatment with 3nM FGF7 resulted in increased levels of Pdx1 transcript (biochem. J.379:749,2004 (J.Biochem., Vol.379, p.749, 2004)).

Shiraki et al investigated the effect of growth factors that specifically enhance differentiation of embryonic stem cells into PDX1 positive cells. They observed that TGF-. beta.2 reproducibly produced a higher proportion of PDX1 positive cells (Genes cells.2005Jun; 10(6):503-16 (Gene to cells; 6.2005, vol.10, 6, p.503-516)).

Gordon et al demonstrated the induction of brachyury [ positive ]/HNF3 [ positive ] endoderm cells from mouse embryonic stem cells in the absence of serum and in the presence of activin in conjunction with inhibitors of Wnt signaling (US 2006/0003446A 1).

Gordon et al (PNAS, Vol 103, page 16806,2006 (Proc. Natl. Acad. Sci. USA, Vol. 103, p. 16806, 2006)) suggested that "production of prepro-bars needs to be accompanied by both Wnt and TGF-. beta./nodal/activin signals".

However, mouse models of embryonic stem cell development may not fully mimic the developmental program in higher mammals (e.g., humans).

Thomson et al isolated embryonic stem cells from human blastocysts (Science 282:114,1998 (Science 282, page 114, 1998)). Meanwhile, Gearhart and colleagues derived a human embryonic germ (hEG) cell line from fetal gonad tissue (Shamblott et al, Proc. Natl. Acad. Sci. USA 95:13726,1998(Shamblott et al, Proc. Natl. Acad. Sci. USA 95, vol. 95, p. 13726, 1998)). Unlike mouse embryonic stem cells, which can be prevented from differentiating simply by culturing with Leukemia Inhibitory Factor (LIF), human embryonic stem cells must be maintained under very specific conditions (U.S. Pat. No.6,200,806; WO 99/20741; WO 01/51616).

D' Amour et al describe the production of enriched cultures of human embryonic stem cell-derived definitive endoderm in the presence of high concentrations of activin and low concentrations of serum (Nature Biotechnology 2005 (Nature Biotechnology, 2005)). Transplantation of these cells under the kidney capsule of mice resulted in differentiation into more mature cells with characteristics of certain endodermal organs. After FGF-10 was added, human embryonic stem cell-derived definitive endoderm cells could be further differentiated into PDX1 positive cells (US 2005/0266554a 1).

D' Amour et al (Nature Biotechnology-24, 1392-: "we have developed a differentiation method that can transform human embryonic stem (hES) cells into cells capable of synthesizing pancreatic hormone: insulin, glucagon, somatostatin, pancreatic polypeptide, and ghrelin (ghrelin). The method mimics pancreatic organogenesis in vivo by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursors to cells capable of expressing endocrine hormones ".

As another example, Fisk et al reported a system for generating islet cells from human embryonic stem cells (US2006/0040387A 1). In this case, the differentiation pathway is divided into three stages. Human embryonic stem cells were first differentiated into endodermal cells using a mixture of sodium butyrate and activin a. The cells are then cultured with a TGF- β antagonist such as a mixture of noggin and EGF or betacellulin to produce PDX1 positive cells. Terminal differentiation was induced by nicotinamide.

In one example, Benvenistry et al states that: "we conclude that: overexpression of PDX1 enhances the expression of pancreatic abundant genes, inducing insulin expression may require an additional signal that is only present in vivo "(Benveniastry et al, Stem Cells 2006; 24:1923 & 1930 (Benveniastry et al, Stem Cells 2006, Vol. 24, p. 1923 & 1930)).

In another example, Grapin-Botton et al states that: early activation of "Ngn 3 almost completely induces glucagon + cells while depleting the pool of pancreatic progenitor cells. Starting from El 1.5, PDX-1 progenitor cells have the ability to differentiate into insulin [ positive ] and PP [ positive ] cells (Johansson KA et al, development Cell 12,457-465, March 2007(Johansson KA et al, Developmental cells, volume 12,457-465, 3 months 2007)).

For example, Diez et al claims; "at 9 and 10 weeks, most glucagon positive cells co-expressed insulin, although clearly cells expressing insulin alone were detected at these stages. Cells co-expressing insulin and glucagon were observed throughout the study (9 to 21 weeks), but they represented only a small fraction of total cells expressing insulin and glucagon "(J Histochem Cytochem. 2009Sep; 57(9):811-24.2009Apr 13 (J. histochemistry and cell chemistry, 2009, 9; 57, 9, 811, 824, 2009, 13).

In one example, "(-) -indoctam V- [ (ILV) ] activates protein kinase C signaling and directs the action of pancreatic-specific ILV and retinoic acid of hESCs that have committed to the endodermal lineage to function by a related mechanism ILV appears to induce PDX-1 expressing cells (percentage of PDX-1 expressing cells)" more strongly than retinoic acid (Nature Chemical Biology 5,195-196(April 2009) ("Nature Chemicals", Vol. 5, p. 195-196 (2009-4 months)).

Lyttle et al claim: "NKX 6-1 co-localized only with insulin cells, indicating that NKX6-1 was only involved in human beta cell development" (Diabetologia 2008Jul:51(7): 1169-.

Therefore, there remains a particular need to develop in vitro methods for producing functional cells expressing insulin that more closely resemble beta cells. The present invention takes the alternative approach of increasing the efficiency of differentiating human embryonic stem cells into insulin expressing cells by generating a cell population that expresses markers characteristic of the pancreatic endoderm lineage, wherein more than 50% of the cells in the cell population co-express PDX-1 and NKX 6.1.

Disclosure of Invention

In one embodiment, the invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, wherein greater than 50% of the cells in the population co-express PDX1 and NKX 6.1.

In one embodiment, the present invention provides a method of differentiating a population of pluripotent stem cells into a population of cells expressing markers characteristic of the pancreatic endocrine lineage, the method comprising the steps of:

a. culturing a population of pluripotent stem cells,

b. differentiating the population of pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage, and

c. treatment of a population of cells expressing markers characteristic of the definitive endoderm lineage with a culture medium supplemented with an activator of protein kinase C can differentiate a population of cells expressing markers characteristic of the definitive endoderm lineage into a population of cells expressing markers characteristic of the pancreatic endoderm lineage.

In one embodiment, greater than 50% of the cells in the population of cells expressing markers characteristic of the pancreatic endoderm lineage produced by the methods of the invention co-express PDX-1 and NKX 6.1.

Drawings

FIGS. 1A and 1B show the expression of PDX1, NKX6.1, and ISL-1 at day 4 of the differentiation protocol stage 4 outlined in example 1. FIG. 1A shows the expression of PDX1 and NKX 6.1. FIG. 1B shows the expression of NKX6.1 and ISL-1.

Fig. 2A and 2B show the effect of PKC activator treatment on the percentage of cells expressing PDX1, NKX6.1, and CDX2 (analyzed by IN Cell Analyzer 1000, fig. 2A), and compare different PKC activators and their effect on the percentage of cells expressing PDX1 and NKX6.1 (analyzed by IN Cell Analyzer, fig. 2B).

FIGS. 3A, 3B and 3C show the circulating C-peptide from SCID-beige mice implanted with cells of the invention under the renal capsule (FIG. 3A) and subcutaneously implanted with a Theracyte device (FIG. 3B). C-peptide levels were measured at the indicated times. FIG. 3C shows a comparison of the C-peptide levels observed after 12 weeks of transplantation in the group implanted with cells under the renal capsule and in the group implanted with cells using a subcutaneous Theracyte device.

FIGS. 4A, 4B, 4C and 4D show the effect of PKC activator treatment on PDX1, NKX6.1, NGN3 and PTF1 α expression in cells treated according to the method described in example 3.

FIGS. 5A, 5B, 5C and 5D show the effect of FGF7 on the expression of NKX6.1, PDX1, PTF 1a and CDX2 in cells treated according to the method described in example 4.

Detailed Description

In order to clearly illustrate this disclosure in a non-limiting manner, embodiments of the invention are divided into the following sections which describe or illustrate certain features, embodiments or applications of the invention.

Definition of

Stem cells are undifferentiated cells defined by their ability to both self-renew and differentiate at the single cell level to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to: the ability to differentiate in vitro into functional cells of multiple cell lineages from multiple germ layers (endoderm, mesoderm, and ectoderm), as well as the ability to produce tissue of multiple germ layers after transplantation and the ability to contribute substantially to most if not all tissues after injection into blastocysts.

Stem cells are classified according to their developmental potential as: (1) totipotency, meaning the ability to produce all embryonic and extra-embryonic cell types; (2) pluripotency, meaning the ability to produce all embryonic cell types; (3) multipotentiality, means capable of producing a subset of cell lines, but all within a particular tissue, organ or physiological system (e.g., Hematopoietic Stem Cells (HSCs) can produce progeny that include (self-renewing) HSCs, blood cell-restricted oligopotent progenitors, and all cell types and elements that are normal components of the blood (e.g., platelets), (4) oligopotency, means capable of producing a subset of cell lines that are more restricted than pluripotent stem cells, and (5) unipotent, means capable of producing a single cell lineage (e.g., spermatogenic stem cells).

Differentiation is the process by which an untargeted ("untargeted") or under-specialized cell acquires the characteristics of a specialized cell (e.g., a nerve cell or muscle cell). Differentiated cells or cells that induce differentiation are cells that have occupied more specialized ("committed") locations in the cell lineage. The term "committed", when applied to the process of differentiation, refers to cells that have progressed to such an extent in the differentiation pathway: under normal circumstances, it will continue to differentiate into a particular cell type or subset of cell types, and under normal circumstances cannot differentiate into another cell type or revert to an under-differentiated cell type. Dedifferentiation refers to the process by which cells revert to an underspecified (or committed) position within the lineage of the cell. As used herein, "lineage of a cell" defines the genetic relationship of a cell, i.e., from which cells it originates and what cells it can produce. Cell lineages locate cells within a genetic program of development and differentiation. Lineage specific markers refer to features that are unambiguously associated with the phenotype of cells of the lineage of interest, and can be used to assess differentiation of non-committed cells to the lineage of interest.

As used herein, "cells expressing markers characteristic of the definitive endoderm lineage" or "stage 1 cells" or "stage 1" refers to cells expressing at least one of the following markers: SOX17, GATA4, HNF3 β, GSC, CER1, Nodal, FGF8, brachyury, Mix-like homeobox protein, FGF4CD48, degermed protein (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, or OTX 2. Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cells and definitive endoderm cells.

As used herein, "cells expressing markers characteristic of the pancreatic endoderm lineage" refers to cells expressing at least one of the following markers: PDX1, NKX6.1, HNF1 β, PTF1 α, HNF6, HNF4 α, SOX9, HB9, or PROX 1. Cells expressing markers characteristic of the pancreatic endoderm lineage include pancreatic endoderm cells, primitive gut tube cells, and posterior foregut cells.

As used herein, "definitive endoderm" refers to cells that have the characteristics of cells produced from the epiblast during gastrulation and form the gastrointestinal tract and its derivatives. Definitive endoderm cells expressed the following markers: HNF3 beta, GATA4, SOX17, Cerberus, OTX2, goose, C-Kit, CD99 and MIXL 1.

As used herein, a "marker" is a nucleic acid or polypeptide molecule that is differentially expressed in a cell of interest. In this context, differential expression means that the level of positive marker is increased, while the level of negative marker is decreased. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cell of interest than in the other cells such that the cell of interest can be identified and distinguished from the other cells using any of a variety of methods known in the art.

As used herein, "pancreatic endocrine cells" or "pancreatic hormone-expressing cells" or "cells expressing markers characteristic of the pancreatic endocrine lineage" refer to cells capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin and pancreatic polypeptide.

Isolation, expansion and culture of pluripotent stem cells

Characterization of pluripotent Stem cells

Pluripotent stem cells can express one or more stage-specific embryonic antigens (SSEA)3 and 4, as well as markers that can be detected using antibodies known as Tra-1-60 and Tra-1-81 (Thomson et al, Science 282:1145,1998(Thomson et al, Science 282, page 1145, 1998)). The differentiation of pluripotent stem cells in vitro results in the loss of expression of SSEA-4, Tra 1-60 and Tra1-81 (if present) and an increase in expression of SSEA-1. Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.). Undifferentiated pluripotent stem cells also typically express OCT4 and TERT, which can be detected by RT-PCR.

Another desirable phenotype of proliferating pluripotent stem cells is the potential to differentiate into cells of all three germ layers: cells of endodermal, mesodermal and ectodermal tissues. The pluripotency of pluripotent stem cells can be confirmed, for example, by: cells were injected into Severe Combined Immunodeficiency (SCID) mice, the teratomas formed were fixed with 4% paraformaldehyde, and then they were examined histologically to determine the presence of cell types from three germ layers. Alternatively, pluripotency can be determined by generating an embryoid body and assessing the presence of markers associated with three germ layers in the embryoid body.

The karyotype of the expanded pluripotent stem cell line can be analyzed using standard G-band techniques and compared to published karyotypes of the corresponding primate species. It is desirable to obtain cells with a "normal karyotype," which means that the cells are euploid, with all human chromosomes present and not significantly altered.

Sources of pluripotent stem cells

The types of pluripotent stem cells that can be used include established pluripotent cell lines derived from tissues formed after pregnancy, including pre-embryonic tissue (e.g., blastocysts), embryonic tissue, or fetal tissue taken at any time during pregnancy, typically, but not necessarily, about 10 to 12 weeks prior to pregnancy. Non-limiting examples are established human embryonic stem cell lines or human embryonic germ cell lines, such as human embryonic stem cell lines H1, H7, and H9 (WiCell). It is also contemplated that the compositions of the present disclosure will be used during initial establishment or stabilization of such cells, in which case the source cells will be primary pluripotent cells taken directly from the source tissue. Also suitable are cells taken from a pluripotent stem cell population that has been cultured in the absence of feeder cells. Also suitable are mutant human embryonic stem cell lines, such as BG01v (BresaGen, Athens, Georgia).

In one example, human embryonic stem cells are prepared as described by Thomson et al (U.S. Pat. No.5,843,780; Science 282:1145,1998 (Science 282, vol. 1145, 1998); curr. Top. Dev. biol.38:133ff.,1998 (Current protocols of developmental biology, vol. 38, vol. 133ff., 1998); Proc. Natl. Acad. Sci. U.S. A.92:7844,1995 (Proc. Natl. Acad. Sci. U.S. 92, Vol. 92, p. 7844, 1995)).

Culture of pluripotent stem cells

In one example, pluripotent stem cells are cultured on a layer of feeder cells that can support the pluripotent stem cells in a variety of ways. Alternatively, pluripotent stem cells are cultured in a culture system that is substantially free of feeder cells, but that also supports proliferation of pluripotent stem cells without substantial differentiation. A medium conditioned by culturing another cell type previously is used to support the growth of pluripotent stem cells in feeder cells-free culture without differentiation. Alternatively, a chemically-defined medium is used to support the growth of pluripotent stem cells in feeder cells-free culture without differentiation.

In one example, pluripotent stem cells can be cultured on a mouse embryonic fibroblast feeder cell layer according to the methods disclosed in the following documents: reubinoff et al (Nature Biotechnology 18: 399-. Alternatively, pluripotent stem cells can be cultured on a mouse embryonic fibroblast feeder cell layer according to the methods disclosed in the following documents: thompson et al (Science 6November 1998: Vol.282.no.5391, pp.1145-1147 (Science, 6.11.1998, 282, 5391, 1145-1147)). Alternatively, pluripotent stem cells can be cultured on any of the feeder cell layers disclosed in the following references: richards et al (Stem Cells 21: 546) -556,2003 (Stem Cells 21, pp. 546-556, 2003)).

In one example, pluripotent stem cells can be cultured on a layer of human feeder cells according to the methods disclosed in the following documents: wang et al (Stem Cells 23:1221-1227,2005 (Stem Cells, Vol. 23, p. 1221-1227, 2005)). In an alternative embodiment, pluripotent stem cells may be cultured on a layer of human feeder cells as disclosed in: stojkovic et al (Stem Cells 200523:306-314,2005 (Stem Cells, Vol. 23, p. 306-314, 2005)). Alternatively, pluripotent stem cells can be cultured on a layer of human feeder cells as disclosed in: miyamoto et al (Stem Cells 22:433-440, 2004) (Stem Cells 22, 433-440). Alternatively, pluripotent stem cells can be cultured on a layer of human feeder cells as disclosed in: amit et al (biol. reprod 68: 2150) -2156,2003 (biol., vol. 68, p. 2150-2156, 2003)). Alternatively, pluripotent stem cells can be cultured on a layer of human feeder cells as disclosed in: inzunza et al (Stem Cells 23:544-549,2005 (Stem Cells 23, vol. 544-549, 2005)).

In one example, pluripotent stem cells may be cultured in a medium obtained according to the method disclosed in US 20020072117. Alternatively, pluripotent stem cells may be cultured in a medium obtained according to the method disclosed in US 6642048. Alternatively, pluripotent stem cells can be cultured in a medium obtained according to the method disclosed in W02005014799. Alternatively, pluripotent stem cells may be cultured in a medium obtained according to the methods disclosed in the following documents: xu et al (Stem Cells 22:972-980,2004 (Stem Cells, Vol. 22, pp. 972-980, 2004)). Alternatively, pluripotent stem cells can be cultured in a medium obtained according to the method disclosed in US 20070010011. Alternatively, pluripotent stem cells may be cultured in a medium obtained according to the method disclosed in US 20050233446. Alternatively, pluripotent stem cells may be cultured in a medium obtained according to the method disclosed in US 6800480. Alternatively, pluripotent stem cells can be cultured in a medium obtained according to the method disclosed in W02005065354.

In one example, pluripotent stem cells can be cultured according to the methods disclosed in: cheon et al (BioReprod DOI: 10.1095/bioleprod.105.046870, October 19,2005 (BioProtobiol, DOI: 10.1095/bioleprod.105.046870, 10/19/2005)). Alternatively, pluripotent stem cells can be cultured according to the methods disclosed in the following documents: levenstein et al (Stem Cells 24:568-574,2006 (Stem Cells, Vol. 24, p. 568-574, 2006)). Alternatively, pluripotent stem cells may be cultured according to the methods disclosed in US 20050148070. Alternatively, pluripotent stem cells may be cultured according to the methods disclosed in US 20050244962. Alternatively, pluripotent stem cells can be cultured according to the method disclosed in W02005086845.

Pluripotent stem cells can be seeded onto a suitable culture substrate. In one embodiment, a suitable culture substrate is an extracellular matrix component, such as a component derived from basement membrane, or a component that may form part of an adhesion molecule receptor-ligand conjugate. In one embodiment, a suitable culture substrate is

(Bidi corporation (Becton Dickenson)).

Is a soluble preparation from Engelbreth-Holm Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane.

Other extracellular matrix components and component mixtures are suitable as alternatives. Depending on the cell type being expanded, this may include laminin, fibronectin, proteoglycans, entactin, heparan sulfate, and the like, alone or in various combinations.

Pluripotent stem cells can be seeded onto the matrix in a suitable distribution in the presence of a medium that promotes cell survival, proliferation, and maintenance of desired properties. All of these characteristics can benefit from careful consideration of the seeding distribution and can be readily determined by one skilled in the art.

Suitable media can be prepared from, for example, Dulbecco's Modified Eagle's Medium (DMEM), Gibco # 11965-092; knock-out of Dulbecco's modified Eagle's medium, KO DMEM, Gibco # 10829-; ham's F12/50% DMEM basal medium; 200mM L-glutamine, Gibco # 15039-; a non-essential amino acid solution, Gibco 11140-050; beta-mercaptoethanol, Sigma (Sigma) # M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco # 13256-.

Forming cells expressing markers characteristic of the pancreatic endoderm lineage from pluripotent stem cells

In one embodiment, the invention provides a method of producing a population of cells expressing markers characteristic of the pancreatic endoderm lineage from pluripotent stem cells, the method comprising the steps of:

a. culturing a population of pluripotent stem cells,

In one aspect of the invention, the invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, wherein greater than 50% of the population co-express PDX-1 and NKX 6.1. In an alternative embodiment, the invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, wherein greater than 60% of the population co-express PDX-1 and NKX 6.1. In an alternative embodiment, the invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, wherein greater than 70% of the population co-express PDX-1 and NKX 6.1. In an alternative embodiment, the invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, wherein greater than 80% of the population co-express PDX-1 and NKX 6.1. In an alternative embodiment, the invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, wherein greater than 90% of the population co-express PDX-1 and NKX 6.1.

In one aspect of the invention, the population of cells expressing markers characteristic of the pancreatic endoderm lineage can also be treated to form a population of cells expressing markers characteristic of the pancreatic endoderm lineage.

Differentiation efficiency can be determined by exposing the treated cell population to an agent (e.g., an antibody) that specifically recognizes a protein marker expressed by cells expressing a marker characteristic of the desired cell type.

Methods for assessing expression of protein markers and nucleic acid markers in cultured or isolated cells are standard methods in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blotting, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (guide to Molecular Biology, eds. (Ausubel et al, 2001 suppl.)) and immunoassays (e.g., immunohistochemical analysis of sliced material), Western blotting and flow cytometric analysis (FACS) of markers accessible in intact cells) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998) ("guide to antibody technology, Cold Spring Harbor Laboratory Press, N.Y., 1998)).

The characteristics of pluripotent stem cells are well known to those skilled in the art, and other characteristics remain to be distinguished. Pluripotent stem cell markers include, for example, expression of one or more of the following markers: ABCG2, CRIPTO, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTFI, ZFP42, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81.

After treatment of pluripotent stem cells with the methods of the invention, the differentiated cells can be purified by exposing a treated population of cells to a protein marker (such as CXCR4) that specifically recognizes the expression of a marker characteristic of the definitive endoderm lineage.

Pluripotent stem cells suitable for use in the present invention include, for example, human embryonic stem cell line H9(NIH code: WA09), human embryonic stem cell line HI (NIH code: WA01), human embryonic stem cell line H7(NIH code: WA07), and human embryonic stem cell line SA002 (Cellartis, Sweden, Inc., Sweden). Cells expressing at least one of the following markers characteristic of pluripotent cells are also suitable for use in the present invention: ABCG2, CRIPTO, CD9, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTFI, ZFP42, SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81.

Markers characteristic of the definitive endoderm lineage are selected from the group consisting of SOX17, GATA4, HNF3 β, GSC, CER1, Nodal, FGF8, Brachyury, Mix-like homeobox proteins, FGF4, CD48, degermed protein (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99 and OTX 2. Suitable for use in the present invention are cells expressing at least one marker characteristic of the definitive endoderm lineage. In one aspect of the invention, the cells expressing markers characteristic of the definitive endoderm system are primitive streak precursor cells.

In another aspect, the cells expressing markers characteristic of the definitive endoderm lineage are mesendoderm cells. In another aspect, the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells.

Markers characteristic of the pancreatic endoderm lineage are selected from PDX1, NKX6.1, HNF1 β, PTF1 α, HNF6, HNF4 α, SOX9, HB9 and PROX 1. Suitable for use in the present invention are cells expressing at least one marker characteristic of the pancreatic endoderm lineage. In one aspect of the invention, the cells expressing markers characteristic of the pancreatic endoderm are pancreatic endoderm cells.

Markers characteristic of the pancreatic endocrine lineage are selected from the group consisting of NGN3, NEUROD, NKX2.2, PDX1, NKX6.1, PAX4 and PAX 6. In one embodiment, the pancreatic endocrine cells are capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin and pancreatic polypeptide. Suitable for use in the present invention are cells expressing at least one marker characteristic of the pancreatic endocrine lineage. In one aspect of the invention, the cells expressing markers characteristic of the pancreatic endocrine system are pancreatic endocrine cells. The pancreatic endocrine cells can be cells that express pancreatic hormone. Alternatively, the pancreatic endocrine cells can be cells that secrete pancreatic hormone.

In one aspect of the invention, the pancreatic endocrine cells are cells that express markers characteristic of the beta cell lineage. Cells expressing markers characteristic of the beta cell line express PDX1 and at least one of the following transcription factors: NGN3, NKX2.2, NKX6.1, NEUROD, SL1, HNF3 β, MAFA, PAX4 and PAX 6. In one aspect of the invention, the cells expressing markers characteristic of the beta cell lineage are beta cells.

Differentiation of pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage

The formation of cells expressing markers characteristic of the definitive endoderm lineage can be determined by detecting the presence of the markers before or after a particular protocol is performed. Pluripotent stem cells do not typically express such markers. Thus, differentiation of pluripotent cells is detected when the cells begin to express them.

The pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by any method in the art or by any method suggested by the present invention.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D 'Amour et al, Nature Biotechnology 23,1534-1541(2005) (D' Amour et al, Nature Biotechnology, Vol.23, p.1534-1541, 2005).

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in Shinozaki et al, Development 131, 1651-.

For example, pluripotent Stem Cells can be differentiated into Cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in McLean et al, Stem Cells 25,29-38(2007) (McLean et al, Stem Cells, Vol. 25, pp. 29-38, 2007).

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the method disclosed in D 'Amour et al, Nature Biotechnology 24,1392-1401(2006) (D' Amour et al, Nature Biotechnology, Vol.24, p.1392-1401, 2006).

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the method disclosed in U.S. patent application No.11/736,908.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the method disclosed in U.S. patent application No.11/779,311.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the method disclosed in U.S. patent application No.60/990,529.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in U.S. patent application No.61/076,889.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the method disclosed in U.S. patent application No.61/076,900.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the method disclosed in U.S. patent application No.61/076,908.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the method disclosed in U.S. patent application No.61/076,915.

Cells expressing markers characteristic of the definitive endoderm lineage to cells expressing markers characteristic of the pancreatic endoderm lineage Differentiation of cells of (2)

a. culturing a population of pluripotent stem cells,

In one embodiment, the protein kinase C activator is selected from the group consisting of (2S,5S) - (E, E) -8- (5- (4- (trifluoromethyl) phenyl) -2, 4-pentadienoylamino) benzolactam, Indolactam V (ILV), phorbol-12-myristate-13-acetate (PMA), and phorbol 12, 13-dibutyrate (PDBu). In one embodiment, the protein kinase C activator is (2S,5S) - (E, E) -8- (5- (4- (trifluoromethyl) phenyl) -2, 4-pentadienoylamino) benzolactam. (2S,5S) - (E, E) -8- (5- (4- (trifluoromethyl) phenyl) -2, 4-pentadienoylamino) benzolactam can be used at a concentration of about 20nM to about 500 nM. (2S,5S) - (E, E) -8- (5- (4- (trifluoromethyl) phenyl) -2, 4-pentadienoylamino) benzolactam is referred to herein as "TPB".

In one embodiment, the protein kinase C activator is supplemented with at least one additional factor selected from the group consisting of: a factor capable of inhibiting BMP, an inhibitor of TGF β receptor signalling, and a Fibroblast Growth Factor (FGF).

In one embodiment, the factor capable of inhibiting BMP is noggin. Noggin may be used at a concentration of about 50ng/ml to about 500. mu.g/ml. In one embodiment, noggin is used at a concentration of 100 ng/ml.

In one embodiment, the TGF β receptor signaling inhibitor is an inhibitor of ALK 5. In one embodiment, the inhibitor of ALK5 is ALK5 inhibitor II. ALK5 inhibitor II may be used at a concentration of about 0.1 μ Μ to about 10 μ Μ. In one embodiment, ALK5 inhibitor II is used at a concentration of 1 μ Μ.

In one embodiment, the fibroblast growth factor is FGF 7. In an alternative embodiment, the fibroblast growth factor is FGF 10.

In one embodiment, fibroblast growth factor may be used at a concentration of about 50pg/ml to about 50 μ g/ml. In one embodiment, fibroblast growth factor is used at a concentration of 50 ng/ml.

Differentiating cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers of the pancreatic endocrine lineage Of (2) cells

In one embodiment, the population of cells expressing markers characteristic of the pancreatic endoderm lineage can be further differentiated into a population of cells expressing markers characteristic of the pancreatic endocrine lineage using any method known in the art.

For example, a population of cells expressing markers characteristic of the pancreatic endoderm lineage can be treated to further differentiate into a population of cells expressing markers characteristic of the pancreatic endocrine lineage according to the methods disclosed in the following documents: d 'Amour et al, Nature Biotechnology,2006 (D' Amour et al, Nature Biotechnology, 2006).

For example, a population of cells expressing markers characteristic of the pancreatic endoderm lineage can be treated according to the methods disclosed in U.S. patent application No.11/736,908 to further differentiate the population of cells expressing markers characteristic of the pancreatic endoderm lineage into a population of cells expressing markers characteristic of the pancreatic endocrine lineage.

For example, a population of cells expressing markers characteristic of the pancreatic endoderm lineage can be treated according to the methods disclosed in U.S. patent application No.11/779,311 to further differentiate the population of cells expressing markers characteristic of the pancreatic endoderm lineage into a population of cells expressing markers characteristic of the pancreatic endocrine lineage.

For example, a population of cells expressing markers characteristic of the pancreatic endoderm lineage can be treated according to the methods disclosed in U.S. patent application No.60/953,178 to further differentiate the population of cells expressing markers characteristic of the pancreatic endoderm lineage into a population of cells expressing markers characteristic of the pancreatic endocrine lineage.

For example, a population of cells expressing markers characteristic of the pancreatic endoderm lineage can be treated according to the methods disclosed in U.S. patent application No.60/990,529 to further differentiate the population of cells expressing markers characteristic of the pancreatic endoderm lineage into a population of cells expressing markers characteristic of the pancreatic endocrine lineage.

For example, a population of cells expressing markers characteristic of the pancreatic endoderm lineage can be treated according to the methods disclosed in U.S. patent application No.61/289,671 to further differentiate the population of cells expressing markers characteristic of the pancreatic endoderm lineage into a population of cells expressing markers characteristic of the pancreatic endocrine lineage.

The invention is further illustrated by, but not limited to, the following examples.

Examples of the invention

Example 1

Form the cell population of the invention

Cells of the human embryonic stem cell line Hl

(1:30 dilutions) (BD Biosciences; Cat. No. 356231) on plates coated with RPMI Medium (Invitrogen; Cat. No.: 22400) + 0.2% FBS +100ng/ml activin A (PeproTech; Cat. No. 120-14) +20ng/ml WNT-3a (Andy Biotech (R)&D Systems); catalog No. 1324-WN/CF), followed by two additional days of treatment with RPMI medium supplemented with 0.5% FBS and 100ng/mL activin a (stage 1), followed by,

DMEM/F12+ 2% FBS +50ng/ml FGF7 for three days (stage 2), followed by

DMEM-high glucose + 1% B27+0.25 μm cyclopamine-KAAD +2 μm retinoic acid

(RA) +100ng/ml noggin for four days (stage 3), followed by either

c. Treatment 1 was carried out: DMEM-high glucose + 1% B27 for four days (phase 4-basal Medium-BM), or

d. Treatment 2 is carried out: DMEM-high glucose + 1% B27+100ng/ml noggin +1 μm ALK5 inhibitor II for four days (stage 4), or

e. Treatment 3 is carried out: DMEM-high glucose + 1% B27+100ng/ml noggin +1 μm ALK5 inhibitor II +20nM phorbol 12, 13-dibutyrate (PDBu) (Calbiochem, Cat. No. 524390) for four days (stage 4).

Cultures were sampled IN duplicate on stage 4 day 4 and then imaged using IN Cell Analyzer 1000(GE Healthcare). Images were taken from 100 fields per well to compensate for any cell loss during the bioassay and subsequent staining. Measurements of total Cell number, total cells expressing PDX1, total cells expressing NKX6.1 and total cells expressing CDX-2 were obtained from each well using IN Cell Developer kit 1.7(GE Healthcare) software. The mean and standard deviation of each duplicate data set was calculated. The total cells expressing PDX1, NKX6.1 and CDX-2 will be reported as a percentage of the total cell population.

As shown in fig. 2A, at the end of phase 4 day 4, about 92% ± 4% of the cells in the cell population expressed PDX1 in all experimental cell populations. However, PDBu (a protein kinase C activator) treatment caused a significant increase in the percentage of NKX 6.1-expressing cells in PDX 1-expressing cell populations compared to cell populations treated with minimal medium (treatment 1) or treated with ALK5 inhibitor II and noggin (treatment 2) (fig. 2A). In the PDBu-treated group, NKX6.1 was expressed in 88% + -4.2% of the total cell population, NKX6.1 was expressed in 62% + -8% of the cells receiving treatment 2, and NKX6.1 was expressed in 46.7 + -0.2% of the cells receiving treatment 1. Most NKX 6.1-expressing cells also express PDX1 in stage 4. These observations were confirmed by overlaying PDX1 expression and NKX6.1 expression images obtained from a given cell population (fig. 1A). These data indicate that treatment of cells with medium supplemented with protein kinase C activator increases the percentage of cells co-expressing PDX1 and NKX6.1 in cell populations expressing markers characteristic of the pancreatic endoderm lineage.

Of the cell

population receiving treatment

1, 10% of the cells expressed CDX2 (intestinal marker) (fig. 2A). Less than 5% of the cell populations receiving treatment 2 or 3 expressed CDX 2. In any case, most cells expressing CDX2 do not co-express PDX1 and NKX 6.1.

Stage 3 parallel cell populations were treated with the following protein kinase C activators: phorbol-12-myristate-13-acetate (PMA) (Calbiochem #524400) at a concentration of 20nM, or [ (2S,5S) - (E, E) -8- (5- (4- (trifluoromethyl) phenyl) -2, 4-pentadienoylamino) benzolactam ] (TPB) (Calbiochem #565740) at a concentration of 50nM, instead of PDBu in treatment 3 above. At the end of day 4 of phase 4, 91% of the cells in the PMA-treated cell population expressed NKX6.1 and 90% of the cells in the TPB-treated cell population expressed NKX 6.1. No significant difference in total cells expressing PDX1 was observed in all treatments. See fig. 2B.

This example demonstrates that protein kinase C activators can be used at relatively low concentrations in order to up-regulate NKX6.1 expression and increase the percentage of cells co-expressing PDX1 and NKX6.1 within a population of cells expressing markers characteristic of the pancreatic endoderm lineage.

Example 2

The invention relates to a cell implantation severe coupletImmunodeficient (SCID) -beige (Bg) mice

Cells of the human embryonic stem cell line Hl

(1:30 dilution) the coated plates were incubated with RPMI medium + 0.2% FBS +100ng/mL activin A +20ng/mL WNT-3a for 1 day, followed by two additional days of treatment with RPMI medium + 0.5% FBS +100ng/mL activin A (stage 1), followed by,

DMEM/F12+ 2% FBS +50ng/ml FGF7 for three days (stage 2), then

DMEM-high glucose + 1% B27+0.25 μm cyclopamine-KAAD +2 μm Retinoic Acid (RA) +100ng/ml noggin for four days (stage 3), followed by

Dmem-high glucose + 1% B27+100ng/ml noggin +1 μ Μ ALK5 inhibitor II +50nM TPB was treated for four days (stage 4).

Male scid-beige mice (C.B-Igh-Ib/GbmsTac-Prkdc) of five to six weeks of age were purchased from Taconic Farms (Taconic Farms)^scid-Lyst^bgN7). Mice were housed in a micro-isolation cage (microisolator cage) with free access to sterile food and water. In preparation for surgery, mice were identified by ear tagging, weighed, and their blood glucose was measured using a handheld glucometer (One Touch, LifeScan).

Mice were anesthetized with a mixture of isoflurane and oxygen, and the surgical site was shaved with small animal scissors. Buprenorphine (Buprenex) was administered subcutaneously to mice at 0.1mg prior to surgery. Successive washes were performed with 70% isopropyl alcohol and 10% povidone-iodine to prepare the surgical site.

The cells at the end of phase 4 were mechanically scraped using a 1ml glass pipette and subsequently transferred to a non-stick plate for overnight culture. During pre-operative preparation of mice, these cells were centrifuged in a 1.5mL centrifuge tube, removing most of the supernatant, leaving just enough to collect the cell pellet. Cells were collected into a ryinin (Rainin) Pos-D positive displacement pipette and the pipette was inverted to allow the cells to settle by gravity. Excess medium was discarded, leaving the concentrated cell preparation for transplantation.

For transplantation, 24G is prepared³/₄The "intravenous catheter" is used to penetrate the renal capsule and remove the needle. The catheter is then advanced under the renal capsule to the far extreme of the kidney. The Pos-D pipette tip was firmly placed into the well of the catheter, and 5 million cells were dispensed from the pipette through the duct under the renal capsule and delivered to the far end of the kidney. The renal capsule is sealed by cryocautery, returning the kidney to its original anatomical position. In parallel, cell aggregates containing 5 million cells were loaded into a 50 μ l device with a Post-D pipette tip. The 50 μ l device was purchased from TheraCyte, Inc. (Irvine, Calif.). After addition, the device was sealed with a type A medical silicone adhesive (Dow Corning, Cat. No. 129109) and then implanted subcutaneously in SICD/Bg mice (animals Nos. 3 and 4). The muscles were closed by continuous suturing with 5-0vicryl sutures and the skin closed with suture clips. Mice were given 1.0mg.kg of meloxicam (Metacam) subcutaneously after surgery. Mice were removed from anesthesia and allowed to recover completely.

After transplantation, mice were weighed once a week and blood glucose was measured twice a week. At various time intervals after transplantation, mice were given 3g/kg of glucose intraperitoneally and blood was drawn 60 minutes after glucose injection through the retro-orbital sinus into centrifuge tubes containing small amounts of heparin. The blood was centrifuged and the plasma was placed in a second microcentrifuge tube, frozen on dry ice and then stored at-80 ℃ until human c-peptide determination was performed. Human C peptide levels were determined using Mercodia/ALPCO diagnostic (ALPCO Diagnostics) hypersensitivity C peptide ELISA (catalog No. 80-CPTHU-E01, ALPCO diagnostic, NH, new hampshire) according to the manufacturer's instructions.

Human C-peptide was detected simultaneously from the sera of animals from both groups as early as 4 to 6 weeks after implantation of the renal capsule group and the group receiving the Theracyte device, and its content increased with time (fig. 3A and 3B). At the end of the three months, we were able to detect significant amounts of circulating human C-peptide after administration of glucose to all animals in the renal capsule group and the group implanted with the Theracyte device. Three months later, serum levels of glucose-stimulated human C-peptide in the kidney capsule group were 1.7 ± 0.5ng/ml (n ═ 4), and serum levels of glucose-stimulated human C-peptide in mice transplanted with the Theracyte device were 1 ± 0.5ng/ml (n ═ 2) (fig. 3C).

This example demonstrates that a cell population co-expressing PDX1 and NKX6.1, generated by a protein kinase C activator, has the ability to differentiate further into insulin-secreting cells in vivo. The potential for further differentiation into insulin-secreting cells is not dependent on the local environment. We demonstrated that co-expressing PDX1 and NKX6.1 cells could further differentiate into insulin secreting cells in the kidney capsule and within the immune protection device at the subcutaneous site.

Example 3

Alternative methods of forming the cell populations of the invention

Cells of human embryonic stem cell line H1

(1:30 Dilute) (BD Biosciences; Cat. No. 356231) on plates coated with RPMI Medium (Invitrogen; Cat. No.: 22400) + 0.2% FBS +100ng/ml activin A (PeproTech; Cat. No. 120-14) +20ng/ml WNT-3a (Andy Biotech (R)&D Systems); catalog No. 1324-WN/CF), followed by two additional days of treatment with RPMI medium supplemented with 0.5% FBS and 100ng/mL activin a (stage 1), followed by,

DMEM/F12+ 2% FBS +50ng/ml FGF7 for three days (stage 2), followed by

DMEM-high glucose + 1% B27+0.25 μm cyclopamine-KAAD +2 μm Retinoic Acid (RA) +100ng/ml noggin for four days (stage 3), followed by either

c. Treatment 4 is carried out: DMEM-high glucose + 1% B27+20nM PDBu +100ng/ml noggin for four days (phase 4), or

d. And (5) carrying out treatment: DMEM-high glucose + 1% B27+100ng/ml noggin +1 μm ALK5 inhibitor II +20nM PDBu for four days (stage 4), or

e. And (6) carrying out treatment: DMEM-high glucose + 1% B27+50ng/ml FGF10+20 nMPBu for four days (stage 4).

The effect of other factors on the increase in the percentage of cells co-expressing PDX1 and NKX6.1 mediated by protein kinase C activators was investigated. Cultures were sampled and image analyzed in duplicate at stage 4 day 4 as described in example 1 above. Expression of ISL1 and NEUROD1 was also recorded.

In this study, most of the cell populations expressing markers characteristic of the pancreatic endoderm lineage were positive for PDX1 expression. Most PDX1 expressing cells were also positive for NKX6.1 expression. As shown in table 1, addition of PKC activator alone favoured the formation of NKX 6.1-expressing cells in the cell population expressing markers characteristic of the pancreatic endoderm lineage (treatment 4). By day 4 of phase 4, 93% of the total cell population were NKX6.1 positive, and almost all NKX6.1 expressing cells were positive for PDX1 expression.

Addition of ALK5 inhibitor II (treatment 5) to the culture medium supplemented with the protein kinase C activator did not affect the observed increase in NKX6.1 expression. 57.1% of the cells in the cell population expressed NEUROD1 and 52.4% of the cells in the cell population expressed ISL1, indicating that the percentage of endocrine precursor cells in the cell population increased after this treatment. See table 1.

PCR analysis of the samples obtained in this example revealed that the cell population receiving treatment 4 had increased expression levels of PDX1, NKX6.1 and PTF1 α compared to the cells receiving treatment 5. See fig. 4A-4D.

On the other hand, a significant increase in the expression of NGN3 was observed in cells receiving ALK5 inhibitor 2 and PDBu (treatment 5). See fig. 4A-4D.

The effect of addition of FGF10 to the culture medium supplemented with protein kinase C activator (treatment 6) was also investigated. Addition of FGF10 at a concentration of 50ng/ml in combination with PDBu (treatment 6) resulted in a cell population expressing markers characteristic of the pancreatic endoderm lineage, 90% of which cells expressed NKX6.1, whereas most NKX 6.1-expressing cells were also CDX2 positive. See table i. The mRNA levels of PDX1, NKX6.1 and PTF1 α were not increased compared to those observed in PDBu and noggin treated cells. See fig. 4A-4D.

This example demonstrates that relatively low concentrations (approximately 20nM) of a protein kinase C activator in combination with a BMP inhibitor can be used to generate a population of cells expressing markers characteristic of the pancreatic endoderm lineage in which greater than 90% of the cells co-express PDX1 and NKX 6.1.

TABLE 1

Example 4

Alternative methods of forming the cell populations of the invention

Cells of the human embryonic stem cell line Hl

DMEM/F12+ 2% FBS +50ng/ml FGF7 for three days (stage 2), followed by either

b. Treatment 7 (T7): DMEM-high glucose + 1% B27+0.25 μm cyclopamine-KAAD +2 μm Retinoic Acid (RA) +100ng/ml noggin for four days (stage 3), or

c. Treatment 8 (T8): DMEM-high glucose + 1% B27+0.25 μm cyclopamine-KAAD +2 μm Retinoic Acid (RA) +100ng/ml noggin + FGF 750 ng/ml treatment for four days (stage 3), followed by either

d. Treatment 9 (T9): DMEM-high glucose + 1% B27+100ng/ml noggin +1 μm ALK5 inhibitor II +20nM PDBu for four days (stage 4), or

e. Treatment 10 (T10): DMEM-high glucose + 1% B27+50ng/ml FGF10+20nM PDBu for four days (phase 4), or

f. Treatment 11 (T11): DMEM-high glucose + 1% B27+20nM PDBu +100ng/ml noggin treatment for four days (phase 4).

Cultures were sampled at stage 4 day 4 IN duplicate and imaged using IN Cell Analyzer 1000(GE Healthcare). Images were taken from 100 fields per well to compensate for any cell loss during the bioassay and subsequent staining. Measurements of total Cell number, total cells expressing PDX1, total cells expressing NKX6.1 and total cells expressing CDX2 were obtained from each well using IN Cell Developer kit 1.7(GE Healthcare) software. The mean and standard deviation of each duplicate data set was calculated. The total cells expressing PDX1, NKX6.1 and CDX-2 will be reported as a percentage of the total cell population.

In the cell population treated with T7 medium followed by T9 medium, approximately 80% of the cells in the cell population expressed NKX 6.1. See table 2. In the cell population treated with T7 medium before T10 medium, 90% of the cells expressed NKX6.1, whereas more CDX2 expressing cells were observed in this treatment. See table 2. Treatment of the cell population with T7 media followed by T11 media produced a cell population that expressed markers characteristic of the pancreatic endoderm lineage, wherein 93% of the cells in the cell population expressed NKX 6.1. Most of the NKX6.1 expressing cells in the cell population also expressed PDX 1.

Cultures treated first with T8 medium and then with T9 medium yielded a population of cells expressing markers characteristic of the pancreatic endoderm lineage, wherein 56.7% of the cells in the population expressed NKX 6.1. See table 2. Cultures treated with T8 media followed by T10 media produced a population of cells expressing markers characteristic of the pancreatic endoderm lineage, in which 63.5% of the cells in the population expressed NKX6.1, and upon which we observed more cells expressing CDX 2. See table 2. Cultures treated first with T8 medium and then with T11 medium yielded a population of cells expressing markers characteristic of the pancreatic endoderm lineage, where 74% of the cells in the population expressed NKX 6.1. See table 2. Most cells expressing NKX6.1 also express PDX 1.

PCR analysis results also supported IN Cell analysis results, i.e., treatment with retinoic acid, cyclopamine, and noggin at stage 3, followed by addition of PKC activator at stage 4, resulting IN increased mRNA levels of NKX6.1 and PTF1 α by stage 4 day 4 (fig. 5A-5D).

TABLE 2

Publications cited throughout this document are hereby incorporated by reference in their entirety. While various aspects of the present invention have been described above with reference to examples and preferred embodiments, it should be understood that the scope of the invention is not limited by the foregoing detailed description, but rather by the claims which follow, appropriately interpreted in accordance with the doctrine of equivalents.

Claims

1. An in vitro method of producing a population of cells expressing pancreatic endoderm, wherein greater than 60% of the cells in the population co-express PDX1 and NKX6.1, comprising the steps of: differentiating a population of definitive endoderm cells in a medium supplemented with an activator of protein kinase C and at least one factor selected from the group consisting of a factor capable of inhibiting BMP, an inhibitor of TGF beta receptor signaling, and a fibroblast growth factor.

2. The method according to claim 1, wherein greater than 70%, 80% or 90% of the cells in said population co-express PDX1 and NKX 6.1.

3. The method according to claim 1 or 2, further comprising the steps of:

a. culturing a population of pluripotent stem cells; and

b. differentiating the population of pluripotent stem cells into a population of definitive endoderm cells.

4. A method according to claim 1 or 2, wherein the protein kinase C activator is selected from the group consisting of (2S,5S) - (E, E) -8- (5- (4- (trifluoromethyl) phenyl) -2, 4-pentadienoylamino) benzolactam, indoctam V, phorbol-12-myristate-13-acetate and phorbol-12, 13-dibutyrate.

5. A method according to claim 1 or 2, wherein the protein kinase C activator is (2S,5S) - (E, E) -8- (5- (4- (trifluoromethyl) phenyl) -2, 4-pentadienoylamino) benzolactam.

6. The method of claim 5, wherein the (2S,5S) - (E, E) -8- (5- (4- (trifluoromethyl) phenyl) -2, 4-pentadienoylamino) benzolactam is used at a concentration of 20nM to 500 nM.

7. The method according to claim 1 or 2, wherein at least one factor selected from the group consisting of: a factor capable of inhibiting BMP and an inhibitor of TGF β receptor signalling.

8. The method of claim 1 or 2, wherein the factor capable of inhibiting BMP is noggin.

9. The method of claim 8, wherein the noggin protein is selected from the group consisting of

The concentration of a.50ng/ml to 500 mug/ml; used at a concentration of 100 ng/ml.

10. The method according to claim 1 or 2, wherein the TGF receptor signalling inhibitor is an ALK5 inhibitor.

11. The method of claim 10, wherein the ALK5 inhibitor is ALK5 inhibitor II.

12. The method of claim 11, wherein ALK5 inhibitor II is administered as

a. Concentration use of 0.1 to 10 μ Μ; or

b. The concentration of 1 μm was used.

13. The method of claim 1 or 2, wherein the fibroblast growth factor is FGF7 or FGF 10.

14. The method of claim 1 or 2, wherein the fibroblast growth factor is selected from the group consisting of

a. Used at a concentration of 50pg/ml to 50 mug/ml; or

b. The concentration of 50ng/ml was used.